The present invention relates to methods of fractionating and collecting analytes. In one embodiment, the invention relates to an improved method for collecting chromatographically separated analytes such as polypeptides, polynucleotides, and polysaccharides.
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Purification and characterization of chemical analytes such as polypeptides, polynucleotides, and polysaccharides, have become increasingly important in the chemical and medical arts. Numerous analytical methods have been developed for a variety of purposes, such as testing for the presence of biological contaminants or toxins, identifying new components in biological systems, and verifying sample purity, for example. Often, analytes of interest are available only in trace amounts or at very low concentrations. Accordingly, there has been much interest in developing analytical techniques with increased sensitivity to facilitate characterization of such analytes.
For many applications, one or more purification steps are necessary before the analyte(s) of interest can be detected or quantified. In the case of analytes which are present in trace amounts, purification has proved difficult for a number of reasons. For example, when analytes elute closely together under given separation conditions, it has been difficult to collect adjacent peaks in a manner that retains resolution, i.e., without significantly diminishing the resolution achieved by the selected purification method. Such small-sample purifications have also been hampered by low sample recoveries due to dilution or adherence of sample on collection vessel surfaces.
Although fraction collection using individual collection vessels has been the traditional mode for collecting and storing resolved sample components, this approach has generally been unsuitable for small sample amounts because of low recoveries as above. Accordingly, other collection methods have been proposed.
One proposed approach involves collecting eluted samples on an adsorbent surface by continuously dragging the outlet of a chromatography column across an adsorbent surface, such that the column effluent is continuously dispensed onto the adsorbent in a continuous trail. Such a method has been proposed by Murata et al. (1993) for collecting polypeptides from a capillary liquid chromatography column. In their method, a pen-holding device is used to maintain the column outlet in continuous contact with a collection membrane.
Although such dragging methods have allowed relatively simple apparatus design, subsequent experience has shown that the fluid outlet often snags on the adsorbent surface, leading to tearing or gouging of the surface or, conversely, locking of the outlet onto the surface so that the outlet cannot move or the adsorbent moves with the outlet. Temporary catching of the outlet on the membrane can lead to discontinuities and other irregularities in the deposited sample, so that the locations of resolved peaks do not correspond with the true elution profile. Tearing or gouging can seriously hinder sample recovery. Long-term locking between the surface and the outlet can result in superimposition of some or all of the resolved peaks, defeating the purpose of the separation. Yet another drawback of the dragging method is the possibility of cross-contamination of eluate due to carry-over of liquid between the capillary outlet and the adsorbent collection layer.
It would be desirable to provide an apparatus and method for collecting small amounts of eluted sample components with high sample recovery, while avoiding the problems mentioned above. In particular, it would be desirable to provide such a method for collecting separated sample components in a manner that allows immediate use or long-term storage for subsequent analysis.
The present invention is directed, in one aspect, to a method of analyzing one or more molecular components in a mixture of components. In the method, a mixture of molecular components, i.e., an analyte mixture, is separated on a capillary liquid chromatography column. The component-containing eluate from the column is deposited as a series of discrete, defined-volume microdrops, along a region of an adsorbent collection layer. During the chromatographic separation, the column eluate may also be monitored to detect the presence of separated components in the eluate. The one or more components deposited in the collection layer are then analyzed by selected analytical techniques.
In one embodiment of the method, the collection layer is immobile during the depositing step, and the depositing step includes reciprocating a deposition head, for depositing the eluate on the collection layer, toward and away from a position of contact with the collection layer, while the deposition head is moved laterally relative to the collection layer. In a preferred embodiment, the deposition head is moved laterally over the collection layer in a linear direction. In an alternative embodiment, the depositing step includes reciprocating a deposition head toward and away from a position of contact with the collection layer while the collection layer is moved laterally relative to the deposition head.
In related aspect, the invention includes a method of collecting one or more molecular components derived from a mixture of components. In the method, a mixture of molecular components is separated on a capillary liquid chromatography column. The component-containing eluate from the column is deposited as a series of discrete, defined-volume microdrops along a region of an adsorbent collection layer. The collected components in the collection layer may then be analyzed by selected analytical techniques.
In another aspect, the invention includes a blotter apparatus which is useful in the methods described above. The apparatus includes an adsorbent collection layer, and means for depositing component-containing eluate from a capillary liquid chromatography column as a series of discrete, defined-volume microdrops, along a region of the adsorbent collection layer. In one embodiment, the apparatus further includes means for monitoring the column eluate to detect the presence of separated components in the eluate, and a control unit operatively connecting the monitoring and depositing means for controlling the flow rate and volume of deposited microdrops. As described above, the collection layer may be immobile, and the depositing means includes a deposition head which is capable of reciprocating toward and away from a position of contact with the collection layer while the deposition head is moved laterally relative to the collection layer. In an alternative embodiment, the deposition is immobile with respect to lateral movement, and the apparatus includes means for moving the deposition layer laterally relative to the deposition head.
The invention also includes a system for analyzing one or more molecular components in a mixture of components. The system includes (i) a capillary liquid chromatography column, (ii) means supplying liquid to the column at a selected flow rate, (iii) means for monitoring the column eluate to detect the presence of separated components in the eluate, (iv) means for depositing component-containing eluate from the column as a series of discrete, defined-volume microdrops, along a region of an adsorbent collection layer, and (v) a control unit operatively connecting the monitoring and depositing means for controlling the flow rate and volume of deposited microdrops.
In one embodiment, the depositing means includes (i) a stage adapted to support the adsorbent collection layer, (ii) a deposition head operable to reciprocate toward and away from a position of contact with the collection layer, and (iii) means for moving the stage and head laterally with respect to one another.
In one embodiment, the stage is effective to hold the collection layer immobile, and the depositing means includes means for moving the deposition head laterally with respect to the collection layer. In an alternative embodiment, the deposition head is immobile with respect to lateral movement, and the system further including means for moving the stage laterally with respect to the deposition head.
The control means may be operable to change the deposition rate and microdrop deposition volume in response to different peak patterns detected by the monitoring means.
These and other objects and features of the invention will be more apparent from the following detailed description when read in light of the accompanying drawings.